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Bearing Mice \ PERGAMON International Journal of Immunopharmacology 10 "0888# 16Ð35 Melatonin administration in tumor!bearing mice "intact and pinealectomized# in relation to stress\ zinc\ thymulin and IL!1 E[ Mocchegiania\\ L[ Perissinb\ L[ Santarellia\ A[ Tibaldia\ S[ Zorzetc\ V[ Rapozzib\ R[ Giacconia\ D[ Buliana\ T[ Giraldic a Immunolo`y Center\ Gerontolo`y Research Department\ Italian National Research Centres on A`in`\"I[N[R[C[A[#\ Ancona\ Italy b Department of Biomedical Sciences and Technolo`ies\ University of Udine\ Udine\ Italy c Department of Biomedical Sciences\ University of Trieste\ Trieste\ Italy Received 10 May 0887^ accepted 09 August 0887 Abstract Melatonin "MEL# may counteract tumors through a direct oncostatic role[ MEL is also an antistress agent with immunoenhancing properties against tumors due to a suppressive role of MEL on corticosterone release[ Rotational stress "RS# "spatial disorientation# facilitates metastasis progression in mice[ Also\ MEL counteracts tumors because of its in~uence on immune responses via the metabolic zinc pool\ which\ is reduced in tumors and stress[ Zinc is required for normal thymic endocrine activity "i[e[ thymulin# and interleukin!1 "IL!1# production[ Because in vivo data is still controversial\ exogenous MEL treatment "11 days in drinking water# in both intact and pinealectomized "px# mice bearing Lewis lung carcinoma leads to signi_cant decrements of metastasis volume\ restoration of the negative crude zinc balance\ recovery of thymulin activity and increment of IL!1 exclusively in intact and px tumor bearing mice subjected to RS[ Signi_cant inverse correlations are found in both stressed intact and px tumor bearing mice after MEL treatment between zinc and corticosterone "r 9[67\ P ³ 9[90^ r 9[79\ P ³ 9[90\ respectively#[ Positive correlations between zinc and IL!1 "r 9[64\ P ³ 9[90^ r 9[62\ P ³ 9[90\ respectively# or thymulin "r 9[64\ P ³ 9[90^ r 9[71\ P ³ 9[90\ respectively# are observed in same models of mice[ These _ndings suggest a MEL action to decrease metastasis mediated by a possible interplay between zinc and MEL\ via corticosterone\ with consequent restoration of thymic e.ciency and IL!1 production[ MEL as an antistress agent with immunoenhancing properties in cancer deserves further consideration[ Þ 0888 International Society for Immunopharmacology[ Published by Elsevier Science Ltd[ Key words] Zinc^ Melatonin^ Stress^ Metastasis^ Thymulin^ Corticosterone^ IL!1 Abbreviations] AAS\ atomic absorption spectrophotometry^ ELISA\ enzyme!linked immunoabsorbent assay^ FTS\ inactive zinc!unbound thymulin^ I\ intact mice^ IL!1\ interleukin!1^ MEL\ melatonin^ MP\ metabolic period^ NF!kB\ Corresponding author[ Tel] ¦28 60 7993005^ Fax ¦28 60 195680^ E!mail] e[mocchegianiÝinrca[it 9081Ð9450:88:,08[99 Þ 0888 International Society for Immunopharmacology[ Published by Elsevier Science Ltd[ PII] S 9 0 8 1 Ð 9 4 5 0 " 8 7 # 9 9 9 5 6 Ð 7 17 E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 nuclear factor!kb^ POMC\ proopiomelanocortin^ Px\ pinealectomized mice^ RIA\ radioimmunoassay^ RS\ rotational stress^ SDI\ stressed intact mice^ SDPx\ stressed pinealectomized mice^ TNF!a\ tumor necrosis factor!a^ ZnFTS\ active zinc!bound thymulin^ ZnFTS¦FTS\ total thymulin[ 0[ Introduction Melatonin "MEL# counteracts tumors with a direct oncostatic role or as an antistress agent with immunoenhancing properties[ In vitro MEL inhibits tumor growth "Shellard\ Whelan + Hill\ 0878^ Cos + Blask\ 0883^ Cos\ Fernandez + Sanchez!Barcelo 0885#[ Leukemia bearing mice have shown an antistress role of MEL with immunoenhancing properties against tumors because of a suppressive action of MEL on corticosterone "Maestroni + Conti\ 0889#[ Rotational Stress "RS# facilitates the progression of tumors in mice\ via neuroendocrine!immune modulation e}ectors of the host "Justice\ 0874^ Vogel + Bower\ 0880^ Giraldi\ Perissin\ Zorzet\ Piccini + Rapozzi\ 0878#[ In turn\ RS a}ects MEL production in BD1F0 mice "Giraldi\ Perissin\ Zorzet + Rapozzi 0883a^ Giraldi\ Perissin\ Zorzet\ Rapozzi + Rodani\ 0883b^ Perissin\ Zorzet\ Rapozzi\ Paoletti + Giraldi\ 0883#\ enhancing circulating MEL in hybrid BD1F0 mice "Perissin\ Zorzet\ Rapozzi + Giraldi\ 0882#\ despite no MEL induction has been reported in C46BL:5 mice "Ebihara\ Marks\ Hudson + Menaker\ 0875#[ MEL action against tumors also occurs because of MEL|s immunomodulatory role "Caroleo\ Frasca\ Nistico| + Doria\ 0881^ Maestroni\ 0882^ Pierpaoli + Regelson\ 0883^ Conti + Maestroni 0884#[ In vivo and in vitro data have recently shown MEL to a}ect thymic endocrine activity "i[e[ thymulin# and interleukin!1 "IL!1#\ in young pinealectomized "px# and old mice by means of metabolic zinc pool turnover\ via glucocorticoids pathway[ The interplay between zinc and MEL\ via corticosterone\ for normal immune e.ciency has assigned to MEL a role as an immunomodulating agent "Mocchegiani et al[\ 0883a\ 0885\ 0887#[ Zinc de_ciency\ impaired thymulin activity and altered serum IL!1 occur both in cancer and stress "Fabris + Mocchegiani\ 0884#[ Zinc is required both for thymulin activity which regulates T!cell mediated immunity "Dardenne et al[\ 0871# and for IL!1 production by lymphocytes "Tanaka\ Shiozawa\ Moromoto + Fujita\ 0889#[ Circulating IL!1 is also a good marker to test immune impairment in zinc!de_cient cancer patients "Prasad\ Beck\ Grabowski\ Kaplan + Plathog\ 0886a#[ With these premises\ MEL administration was carried out in px and intact mice bearing Lewis lung carcinoma subjected or not to RS\ and metastasis volume\ metabolic zinc pool\ thymulin activity\ corticosterone and IL!1 plasma levels were measured in order to further verify MEL|s antistress role with immunoenhancing properties "Maestroni + Conti\ 0889# and:or its direct oncostatic role "Shellard et al[\ 0878^ Cos + Blask\ 0883^ Cos et al[\ 0885#[ MEL action against tumors is still controversial and unde_ned "Stanberry\ Das Gupta\ + Beattie\ 0872^ Blask\ 0873^ Slominski + Pruski\ 0882^ Giraldi et al[\ 0883a\b^ Karasek\ 0886# and requires urgent clari_cation[ 1[ Experimental procedures 1[0[ Animals\ tumor transplantation and measurements Female BD1F0 mice "C46BL:5×DBA:1 F0# "1 months of age# "Charles River\ Calco\ Como\ Italy# weighing 07Ð10 g\ were used[ Lewis lung carcinoma was provided by the National Cancer E[ Mocche`iani et al[:International Journal of Immunopharmacolo`y 10 "0888# 16Ð35 18 Institute\ Bethesda MD\ U[S[A[ The subcutaneous propagation of tumor in BD1F0 mice\ as well as the measure of primary tumor volume and lung metastasis were performed as described elsewhere "Giraldi et al[\ 0878^ Giraldi et al[\ 0883a\b#[ All animals were weighed before the experiment and at sacri_ce to determine whether the e}ects seen were related to dietary intake "Mocchegiani et al[\ 0887#[ Hybrid BD1F0 mice were chosen because the syngenic implantation of Lewis lung carci! noma\ whose malignant progression is sensitive to RS\ works well in this strain "Giraldi et al[\ 0878#[ 1[1[ Animal housing and rotational stress Mice\ with drinking water and pellet food "Nossan\ Italy# ad libitum\ were kept 4 per cage in order to avoid the e}ects of overcrowding or isolation on tumor progression "Riley\ Fitzmaurice + Spackman\ 0870#[ The cages were placed in a low housing stress "protected# environment for 1 weeks before tumor inoculation\ in order to allow the animals to recover from the stress of shipment "Riley et al[\ 0870#[ As such\ mice become adapted to the new housing conditions[ Temperature and humidity were constant "19>C and 59)\ respectively#[ The cycle of illumination was 01:01 h[ "lights on from 9799Ð1999 h# with an intensity of 1999 lux in the cages[ RS "spatial disorientation# was applied to the animals in the low stress environment by spinning the cages at 34 rpm for 09 min every hour from time of tumor inoculation until sacri_ce\ as described elsewhere "Giraldi et al[\ 0878^ 0883b#[ RS is a good model for stress and tumor progression studies in animals "Riley et al[\ 0870^ Justice\ 0874^ Giraldi et al[\ 0878^ 0883a\b#[ 1[2[ Pinealectomy and melatonin administration Surgical pinealectomy was performed using a procedure described previously "Moechegiani et al[\ 0885#[ A post mortem inspection of the brain was performed at sacri_ce in order to exclude in px animals pineal gland presence[ The pinealectomy was performed on mice at 1 months of age[ Tumour inoculation and melatonin treatment were performed 1 months later because 1 months is su.cient time to allow for the disappearance of endogenous nocturnal peak of MEL in inbred mice "Conti + Maestroni 0885#[ MEL "0[14 mg:Kg:night# was administered into the drinking water for 11 days in order to see MEL e}ects on metastasis volume in relation to RS in tumor bearing BD1F0 mice "Giraldi et al[\ 0883a\b#[ MEL "5[1 mg:l# was dissolved in water containing 9[1) ethanol[ The MEL!water was administered during the dark:night period "1999Ð97]99 h#\ from tumor inoculation until sacri_ce[ MEL!containing bottles were removed from 9799Ð1999 h[ Normal tap water was administered during this light:day period[ MEL treatment\ tumor inoculation and rotational stress were carried out in mice at the age of 3 months[ Primary tumor volume was determined on day 03 and lung metastasis were measured at sacri_ce on day 11\ respectively\ from tumor inoculation[ The age of mice "experimental groups and controls# averaged 3[6 months at sacri_ce[ The experimental model was carried out in MayÐJune because of seasonal variations of MEL and the e}ect of RS on metastasis volume
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